Characterization of the StcE protease activity of Escherichia coli O157:H7

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.     

The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen and tightly adheres to human colonic epithelium by forming attaching/effacing lesions. To reach the epithelial surface, EHEC must penetrate the thick mucus layer protecting the colonic epithelium. In this study, we investigated how EHEC interacts with the intestinal mucus layer using mucin‐producing LS174T colon carcinoma cells and human colonic mucosal biopsies. The level of EHEC binding and attaching/effacing lesion formation in LS174T cells was higher compared to mucin‐deficient colon carcinoma cell lines, and initial adherence was independent of the presence of flagellin, Escherichia coli common pilus, or long polar fimbriae. Although EHEC infection did not affect gene expression of secreted mucins, it resulted in reduced MUC2 glycoprotein levels. This effect was dependent on the catalytic activity of the secreted metalloprotease StcE, which reduced the inner mucus layer and thereby promoted EHEC access and binding to the epithelium in vitro and ex vivo. Given the lack of efficient therapies against EHEC infection, StcE may represent a suitable target for future treatment and prevention strategies.     

StcE,a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.     

Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation.     

Connexins in respiratory and gastrointestinal mucosal immunity

The mucosal lining forms the physical and chemical barrier that protects against pathogens and hostile particles and harbors its own population of bacteria, fungi and archea, known as the microbiota. The immune system controls tolerance of this population of microorganisms that have proven to be beneficial for its host. Keeping its physical integrity and a correct balance with the microbiota, the mucosa preserves its homeostasis and its protective function and maintains host’s health. However, in some conditions, pathogens may succeed in breaching mucosal homeostasis and successfully infecting the host. In this review we will discuss the role the mucosa plays in the defense against bacterial pathogens by considering the gap junction protein connexins. We will detail their implication in mucosal homeostasis and upon infection with bacteria in the respiratory and the gastrointestinal tracts.     

Atypical Shigella boydii 13 encodes virulence factors seen in attaching and effacing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes watery diarrhea and hemorrhagic colitis. In this study, we identified StcE, a secreted zinc metalloprotease that contributes to intimate adherence of EHEC to host cells, in culture supernatants of atypical Shigella boydii 13 (Shigella B13) strains. Further examination of the Shigella B13 strains revealed that this cluster of pathogens does not invade but forms pedestals on HEp-2 cells similar to EHEC and enteropathogenic E.?coli. This study also demonstrates that atypical Shigella B13 strains are more closely related to attaching and effacing E.?coli and that their evolution recapitulates the progression from ancestral E.?coli to EHEC.     

Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation,intestinal colonization and colonic hyperplasia in mice

Citrobacter rodentium infection of mice serves as a relevant small animal model to study enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) infections in man. Enteropathogenic E. coli and EHEC translocate Tir into the host cytoplasmic membrane, where it serves as the receptor for the bacterial adhesin intimin and plays a central role in actin condensation beneath the adherent bacterium. In this report, we examined the function of C. rodentium Tir both in vitro and in vivo. Similar to EPEC, C. rodentium Tir is tyrosine phosphorylated and is essential for actin condensation. Citrobacter Tir and EPEC Tir are functionally interchangeable and both require tyrosine phosphorylation to mediate actin rearrangements. In contrast, Citrobacter Tir supports actin nucleation in EHEC independent of tyrosine phosphorylation, while EHEC Tir cannot replace Citrobacter Tir for this function. This indicates that C. rodentium and EPEC use an actin nucleating mechanism different from EHEC. We also found that Tir is expressed and translocated into mouse enterocytes in vivo by C. rodentium during infections. This represents the first direct demonstration of a type III effector translocated in vivo into a natural host by any pathogen. In addition, we showed that Tir, but not its tyrosine phosphorylation, is essential for C. rodentium to colonize the large bowel and induce attaching/effacing (A/E) lesions and colonic hyperplasia in mice, and that both EPEC Tir and EHEC Tir can substitute for Citrobacter Tir for these activities in vivo. These results thus demonstrate that Tir is an essential virulence factor in this infection model. The data also show that the function of Tir tyrosine phosphorylation and its subsequent actin nucleating activity are not essential for C. rodentium colonization of the mouse gut nor for inducing A/E lesions and colonic hyperplasia, thereby uncoupling colonization and disease from actin condensation for this A/E pathogen.     

Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.     

Crystal structure of EHEC intimin: insights into the complementarity between EPEC and EHEC

Enterohaemorrhagic E. coli (EHEC) O157:H7 is a primary food-borne bacterial pathogen capable of causing life-threatening human infections which poses a serious challenge to public health worldwide. Intimin, the bacterial outer-membrane protein, plays a key role in the initiating process of EHEC infection. This activity is dependent upon translocation of the intimin receptor (Tir), the intimin binding partner of the bacteria-encoded host cell surface protein. Intimin has attracted considerable attention due to its potential function as an antibacterial drug target. Here, we report the crystal structure of the Tir-binding domain of intimin (Int188) from E. coli O157:H7 at 2.8 Å resolution, together with a mutant (IntN916Y) at 2.6 Å. We also built the structural model of EHEC intimin-Tir complex and analyzed the key binding residues. It suggested that the binding pattern of intimin and Tir between EHEC and Enteropathogenic E. coli (EPEC) adopt a similar mode and they can complement with each other. Detailed structural comparison indicates that there are four major points of structural variations between EHEC and EPEC intimins: one in Domain I (Ig-like domain), the other three located in Domain II (C-type lectin-like domain). These variations result in different binding affinities. These findings provide structural insight into the binding pattern of intimin to Tir and the molecular mechanism of EHEC O157: H7.     

Interactions with M Cells and Macrophages as Key Steps in the Pathogenesis of Enterohemorragic Escherichia coli Infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer''s patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.     

Tyrosine phosphorylation controls cortactin binding to two enterohaemorrhagic Escherichia coli effectors: Tir and EspFu/TccP

Enterohaemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen that, upon infection, causes destruction of the microvilli brush border of intestinal cells. EHEC is able to recruit several host cell proteins and induce actin accumulation beneath its adherence site, forming a pedestal-like structure upon which the bacterium is firmly attached. Injection of bacterial effectors into the host cells is required to trigger the recruitment and activation of proteins, such as cortactin, neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3 complex, directly involved in the actin polymerization process. We found that cortactin, an actin-binding protein, has a pivotal role during pedestal formation by EHEC. Cortactin was found to bind directly to two important virulence factors of EHEC, Tir and EspF(u), which are translocated into the host cells during infection. Binding of cortactin to these effectors is dependent upon tyrosine phosphorylation and a balance between tyrosine phosphorylation and dephosphorylation of cortactin is required to regulate pedestal formation by EHEC.     

Altering Mucus Rheology to “Solidify” Human Mucus at the Nanoscale

The ability of mucus to function as a protective barrier at mucosal surfaces rests on its viscous and elastic properties, which are not well understood at length scales relevant to pathogens and ultrafine environmental particles. Here we report that fresh, undiluted human cervicovaginal mucus (CVM) transitions from an impermeable elastic barrier to non-adhesive objects sized 1 µm and larger to a highly permeable viscoelastic liquid to non-adhesive objects smaller than 500 nm in diameter. Addition of a nonionic detergent, present in vaginal gels, lubricants and condoms, caused CVM to behave as an impermeable elastic barrier to 200 and 500 nm particles, suggesting that the dissociation of hydrophobically-bundled mucin fibers created a finer elastic mucin mesh. Surprisingly, the macroscopic viscoelasticity, which is critical to proper mucus function, was unchanged. These findings provide important insight into the nanoscale structural and barrier properties of mucus, and how the penetration of foreign particles across mucus might be inhibited.     

Characterization of the NleF effector protein from attaching and effacing bacterial pathogens

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.     

肠出血性大肠杆菌Ⅲ型分泌系统效应因子与宿主细胞相互作用研究进展

肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)通过其Ⅲ型分泌系统将效应因子注入到宿主细胞内,破坏宿主细胞内的多种信号通路从而有利于细菌的感染及定植。近年来对于EHEC Ⅲ型分泌系统效应因子与宿主细胞相互作用研究成为EHEC致病机制研究新的热点,研究表明,除了经典的效应因子外,一些新发现的效应因子在细菌的致病过程中也发挥着重要作用,有些效应因子能够抑制宿主细胞内正常的信号通路,有些效应因子还具有抑制细胞凋亡,干扰炎症信号通路和抑制吞噬的作用。这些发现揭示了EHEC效应因子具有多种功能,它们通过与宿主细胞间的相互作用,在细菌的感染过程中发挥着重要作用。     

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom–up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom–up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.     

Transepithelial neutrophil migration is CXCR1 dependent in vitro and is defective in IL-8 receptor knockout mice

Neutrophil migration across infected mucosal surfaces is chemokine dependent, but the role of chemokine receptors has not been investigated. In this study, chemokine receptors were shown to be expressed by epithelial cells lining the urinary tract, and to play an essential role for neutrophil migration across the mucosal barrier. Uroepithelial CXCR1 and CXCR2 expression was detected in human urinary tract biopsies, and in vitro infection of human uroepithelial cell lines caused a dramatic increase in both receptors. As a consequence, there was higher binding of IL-8 to the cells and the IL-8-dependent neutrophil migration across the infected epithelial cell layers was enhanced. Abs to IL-8 or to the CXCR1 receptor inhibited this increase by 60% (p<0.004), but anti-CXCR2 Abs had no effect, suggesting that CXCR1 was the more essential receptor in this process. Similar observations were made in the mouse urinary tract, where experimental infection stimulated epithelial expression of the murine IL-8 receptor, followed by a rapid flux of neutrophils into the lumen. IL-8 receptor knockout mice, in contrast, failed to express the receptor, their neutrophils were unable to cross the epithelial barrier, and accumulated in massive numbers in the tissues. These results demonstrate that epithelial cells express CXC receptors and that infection increases receptor expression. Furthermore, we show that CXCR1 is required for neutrophil migration across infected epithelial cell layers in vitro, and that the murine IL-8 receptor is needed for neutrophils to cross the infected mucosa of the urinary tract in vivo.     

Role of computer-assisted analysis of the corneal endothelium in vitreoretinal surgery with intraocular silicone oil injection: a technical report

The innermost lining of the cornea consists of a single layer of cells called the endothelium. Despite its name, the endothelium of the cornea differs considerably from the vascular endothelium, both functionally and morphologically. The corneal endothelium plays a fundamental role in maintaining the transparency of the corneal membrane, as the result of both its function as a barrier against penetration of the aqueous humor in the parenchyma and its ability to remove water from the stroma (usually referred to as the endothelial "pump" function). Any abnormality in the corneal endothelium causes, first, the impairment of its function as a barrier and pump due to the loss of stromal anti-turgor mechanisms, followed by edema and possible development into keratopathy. The specular microscope is an instrument which makes it possible to see the endothelial "mosaic" in the reflected image of the posterior corneal surface. A large variety of clinical specular microscopes is presently available, both contact and non-contact, which allow, for easy and rapid photography of the corneal endothelium "in vivo". In the present case, we used a non-contact computerized specular microscope to analyze the corneal endothelium in a group of patients affected by retinal detachment who needed to undergo vitreoretinal surgery with immission of silicone oil into the vitreal chamber.     

The enterohaemorrhagic Escherichia coli (serotype O157:H7) Tir molecule is not functionally interchangeable for its enteropathogenic E. coli (serotype O127:H6) homologue

A major virulence determinant of enteropathogenic Escherichia coli (EPEC) is the Tir molecule that is translocated into the plasma membrane where it orchestrates cytoskeletal rearrangements. Tir undergoes several phosphorylation events within host cells, with modification on a tyrosine essential for its actin-nucleating function. The EHEC (serotype O157:H7) Tir homologue is not tyrosine phosphorylated implying that it uses an alternative mechanism to nucleate actin. This is supported in this study by the demonstration that EHEC Tir is unable to functionally substitute for its EPEC homologue. Like EPEC, the EHEC Tir molecule is phosphorylated within host cells, with the actin-nucleating dysfunction correlated to an altered modification profile. In contrast to EHEC Tir, the EPEC Tir molecule mediated actin nucleation whether delivered into host cells by either strain. Thus, it would appear that EHEC encodes specific factor(s) that facilitate the correct modification of its Tir molecule within host cells. Domain-swapping experiments revealed that the N-terminal, α-actinin binding, Tir domains were functionally interchangeable, with both the actin-nucleating dysfunction and altered modification profiles linked to the EHEC C-terminal Tir domain. This tyrosine-independent modification process presumably confers an advantage to EHEC O157:H7 and may contribute to the prevalence of this strain in EHEC disease. The presented data are also consistent with EPEC and EHEC sharing non-phosphotyrosine phosphorylation event(s), with an important role for such modifications in Tir function. An EHEC-induced phosphotyrosine dephosphorylation activity is also identified.     

肠黏膜屏障与肠道菌群的相互关系

摘要：人类肠道中微生物群与肠道环境相互作用以维持机体健康。肠黏膜屏障主要由黏液层、肠道菌群、肠道免疫系统和肠上皮细胞本身的完整性等构成。肠道作为直接与大量菌群接触的器官,其屏障功能在肠道健康中的作用尤为显著。肠道菌群与肠道屏障相互作用,保持肠道菌群与肠道屏障相对稳定,肠道菌群参与肠道免疫反应的建立,共同建立机体天然防御系统,在保持肠道免疫的动态平衡中具有重要作用。当两者之间的平衡被打破时,可诱发功能性胃肠病（如肠易激综合征）及免疫相关性疾病（如炎症性肠病）。本文主要阐述肠黏膜屏障与肠道菌群之间的相互关系以及与肠道屏障功能障碍相关的肠道疾病。